Quantification Of Neurite Degeneration With Enhanced Accuracy And Efficiency nyob rau hauv Vitro Model Of Parkinson's Disease Part 1

Abstract

Neurite degeneration yog txuam nrog cov theem pib ntawm cov kab mob neurodegenerative xws li Alzheimer's disease, Parkinson's disease (PD), thiab amyotrophic lateral sclerosis. Ib txoj hauv kev uas feem ntau siv los txheeb xyuas cov neurite degeneration suav nrog kev suav ntawm Degeneration Index (DI) tom qab kev siv cov cuab yeej Analyze Particles ntawm ImageJ txhawm rau txheeb xyuas cov kab mob neurite hauv micrographs ntawm cov kab lis kev cai. Txawm li cas los xij, DI kev tshuaj ntsuam xyuas muaj ntau hom kev ntsuas qhov yuam kev, tuaj yeem siv sijhawm los ua, thiab txwv tsis pub siv.

Neurodegeneration yog hais txog kev hloov pauv uas tshwm sim hauv lossis ib puag ncig cov neurons, uas yuav cuam tshuam rau tib neeg kev paub txog kev ua haujlwm, kev txawj ntse, kev xav, kev coj cwj pwm, thiab lwm yam.

Ua ntej, neurodegeneration tsis tas txhais tau hais tias nco poob nrog hnub nyoog. Qee tus neeg muaj peev xwm tuav tau lub cim xeeb zoo thiab tseem tuaj yeem nkag siab txog kev ncaj ncees. Qhov no yog vim tib neeg lub cim xeeb cuam tshuam los ntawm kev sib xyaw ua ke xws li kev tawm dag zog, kev noj haus, thiab kev ua neej.

Qhov thib ob, tib neeg lub hlwb tuaj yeem rov tsim cov kev sib txuas ntawm neural tshiab thiab tsim kev nco tshiab, uas yog hu ua plasticity. Txawm tias muaj hnub nyoog li cas los, lub hlwb tseem kawm tas li thiab tsim kev nco tshiab. Yog li ntawd, neurodegeneration tsuas yog ib qho kev hloov pauv hauv lub hlwb thiab tsis tas txhais tau tias kev nco yuav tsis zoo.

Thaum kawg, kev tswj hwm tus cwj pwm zoo kuj tuaj yeem txhim kho tus neeg nco. Kev txhawj xeeb thiab kev ntxhov siab tuaj yeem muaj kev cuam tshuam tsis zoo rau lub hlwb, ua rau kev nco tsis tau. Tus cwj pwm zoo tuaj yeem txhawb lub hlwb kev noj qab haus huv, thiab txhim kho kev nco thiab kev txawj ntse.

Piv txwv li, neurodegeneration tsis tas yuav ua rau kev nco tsis tau, thiab kev xav zoo rau lub neej tuaj yeem pab peb tswj kev nco zoo thiab kev txawj ntse. Yog li ntawd, peb yuav tsum tswj hwm tus cwj pwm zoo, thiab ntxiv dag zog rau kev tawm dag zog, noj zaub mov, thiab tswj kev ua neej kom muaj lub hlwb noj qab haus huv thiab nco. Nws pom tau tias peb yuav tsum txhim kho peb lub cim xeeb. Cistanche tuaj yeem txhim kho kev nco, vim Cistanche tseem tuaj yeem tswj hwm qhov sib npaug ntawm cov tshuaj neurotransmitters, xws li nce qib ntawm acetylcholine thiab kev loj hlob. Cov khoom no tseem ceeb heev rau kev nco thiab kev kawm. Tsis tas li ntawd, cov nqaij kuj tuaj yeem txhim kho cov ntshav khiav thiab txhawb nqa cov pa oxygen, uas tuaj yeem ua kom lub hlwb tau txais cov khoom noj txaus thiab lub zog, yog li txhim kho qhov tseem ceeb thiab kev ua haujlwm ntawm lub hlwb.

Nyem paub txoj hauv kev los txhim kho lub hlwb

Ntawm no, peb piav qhia txog txoj hauv kev txhim kho rau kev ua DI tshuaj ntsuam xyuas. Qhov tseeb ntawm kev ntsuas tau raug txhim kho los ntawm kev hloov kho cov txheej txheem xaiv rau kev kuaj xyuas cov neurite fragments, tshem tawm cov duab artifacts thiab non-neurite cov ntaub ntawv los ntawm cov duab, thiab optimization ntawm cov duab sib piv. Cov kev txhim kho zoo li no tau muab coj los siv rau hauv ImageJ macro uas ua kom nrawm thiab ua tiav DI tsom xam ntawm ntau cov duab. Cov macro nta kev ua haujlwm rau kev tshem tawm ntawm lub cev ntawm lub cev los ntawm micrographs, yog li nthuav dav daim ntawv thov DI tshuaj ntsuam xyuas los siv hauv cov kev sim cuam tshuam nrog cov kab lis kev cai sib txawv.

Peb nthuav tawm cov kev tshawb nrhiav pom tias, piv nrog rau txoj kev siv cov pa, cov txheej txheem kev tshuaj ntsuam xyuas tau zoo dua los ntsuas qhov tseeb thiab yuav tsum tau siv sijhawm tsawg dua los ua.

Tsis tas li ntawd, peb ua kom pom cov txiaj ntsig ntawm txoj hauv kev los tshawb xyuas neurite degeneration hauv cell kab lis kev cai qauv ntawm PD los ntawm kev ua ib qho kev sim qhia txog cov teebmeem ntawm c-Jun N-terminal kinase (JNK) ntawm neurite degeneration induced los ntawm oxidative kev nyuaj siab nyob rau hauv tib neeg mesencephalic hlwb. Txoj kev txhim kho kev tshuaj ntsuam no yuav raug siv los ua kom tau txais kev pom tshiab rau hauv cov xwm txheej hauv qab neurite degeneration thiab kev loj hlob ntawm cov kab mob neurodegenerative.

Ntsiab lus:

Axonal degeneration; degeneration index; neurite degeneration; neurodegeneration; oxidative kev nyuaj siab; Tus kab mob Parkinson.

Nqe lus tseem ceeb

Neurite degeneration yog ib qho kev tshwm sim ntawm tes nrog rau cov theem pib ntawm ntau hom neurodegenerative mob. Molecular yam uas tswj cov neurite degeneration tseem tsis to taub, thiab cov txheej txheem uas twb muaj lawm rau kev kawm neurite degeneration muaj tsawg daim ntawv thov thiab kev ua tau zoo. Ntawm no, peb txheeb xyuas cov kev hloov kho rau cov txheej txheem siv dav rau kev tshuaj xyuas neurite degeneration uas txo qhov ntsuas qhov yuam kev.

Cov txheej txheem kev txhim kho zoo li no tau muab tso rau hauv ImageJ macro, yog li ua kom yooj yim thiab ua tiav kev soj ntsuam ntawm neurite degeneration siv cov micrographs loj. Siv cov qauv kab lis kev cai ntawm tes ntawm Parkinson's disease (PD), peb pom tau tias qhov macro pab txhawb kev ntsuas qhov tseeb thiab ua tau zoo ntawm neurite degeneration ntsuas thaum nthuav dav qhov tsim nyog ntawm txoj kev tshuaj ntsuam rau kev sim uas cuam tshuam nrog kev coj noj coj ua.

Taw qhia

Neurite degeneration yog cov txheej txheem ntawm tes uas axons lossis dendrites tau ua haujlwm tsis zoo thiab hloov pauv morphologic, ua rau poob ntawm neurite kev ncaj ncees. Neurite degeneration yog cov txheej txheem evolutionarily conserved uas yog ib qho tseem ceeb rau kev tsim kom zoo ntawm neural tes hauj lwm thaum lub sij hawm txoj kev loj hlob ntawm lub hauv paus paj hlwb (Neukomm thiab Freeman, 2014).

Txawm li cas los xij, cov txheej txheem kuj tau tshwm sim los ntawm kev teb rau ntau hom kev raug mob xws li kev puas tsuaj los yog raug rau cov tshuaj siv tshuaj kho mob (Salvadores li al., 2017). Tsis tas li ntawd, neurite degeneration yog txuam nrog kev loj hlob ntawm ntau yam muaj hnub nyoog ntsig txog cov kab mob neurodegenerative, suav nrog Alzheimer's disease, Parkinson's disease (PD), Huntington's disease, thiab amyotrophic lateral sclerosis.

Interestingly, rau tag nrho cov xws li neurodegenerative ntshawv siab, accumulating pov thawj qhia tias neurite degeneration yog ib qho tseem ceeb, theem pib tshwm sim uas precedes neuronal tuag los yog pib ntawm cov tsos mob (Serrano-Pozo li al., 2011; Adalbert thiab Coleman, 2013; Marangoni li al. ., 2014; Suzuki et al., 2020). Yog li, kev tshawb fawb tshawb fawb txog yam uas tswj cov txheej txheem cellular no yuav qhia tawm lub hom phiaj tshiab rau kev kho ntau yam mob neurodegenerative.

Kev tshawb xyuas neurite degeneration yog qhov tseem ceeb tshwj xeeb rau kev nkag siab PD, kab mob tshwm sim los ntawm kev sib sau ntawm cov protein aggregates uas muaj a-synuclein, kev poob ntawm dopaminergic neurons hauv substantia nigra pars compacta (SNpc), thiab kev txhim kho ntau yam ntawm lub cev muaj zog thiab kev txawj ntse. (Aarsland et al., 2017).

Cov ntaub ntawv pov thawj los ntawm kev tshawb fawb tom qab thiab tib neeg cov duab qhia tau hais tias a-synuclein aggregates muaj ntau nyob rau hauv neuronal fibers ntau dua li nyob rau hauv lub cev ntawm tes, thiab degradation ntawm dopaminergic axons tshwm sim ntau dua li poob ntawm dopaminergic neurons hauv SNpc thaum lub sij hawm ua rau kev kho mob. (Tagliaferro thiab Burke, 2016).

Thaum cov kev sib cuam tshuam ntawm cov molecular uas ua rau muaj kev cuam tshuam loj zuj zus ntawm dopaminergic neurites tseem nkag siab tsis tiav, cov pov thawj tsis ntev los no qhia tau hais tias neurite degeneration tuaj yeem kho los ntawm cov xwm txheej ywj pheej ntawm cov teeb liab cascades hauv qab neuronal tuag (Fukui, 2016; Geden thiab Deshmukh, 2016). Yog li, cov kev tshawb fawb uas ntsuas ncaj qha rau lub physiological underpinnings ntawm neurite degeneration yog ib qho tseem ceeb rau txoj kev loj hlob ntawm kev kho mob uas impede thaum ntxov PD kev loj hlob.

Hauv kev teb rau cov pov thawj ntawm lub luag haujlwm tseem ceeb rau neurite degeneration hauv kev loj hlob ntawm cov kab mob neurodegenerative, ntau txoj hauv kev tau tsim los ntsuas neurite degeneration siv micrographs ntawm kab lis kev cai neurons. Piv txwv li, ntau txoj kev sib txawv tau tsim los ntsuas neurite degeneration siv software los kuaj xyuas tus lej neurite tsawg lossis ntev (Ofengeim li al., 2012; Johnstone li al., 2018).

Txawm li cas los xij, txij li cov txheej txheem zoo li no tsis ncaj qha soj ntsuam neurite fragmentation, kev txhais cov txiaj ntsig los txiav txim siab qhov cuam tshuam ntawm qhov sib txawv ntawm neurite degeneration tuaj yeem cuam tshuam los ntawm qhov cuam tshuam ntawm qhov sib txawv ntawm neurite outgrowth. Qee cov kev tshawb fawb pab pawg kuj tau ntsuas neurite degeneration los ntawm kev ntsuas cov duab ntawm cov neurites raws li cov yam ntxwv ntawm morphologic thiab cov degree ntawm pom kev tawg (Zhai li al., 2003; Nakamura li al., 2016; Krishtal et al., 2017). Txawm li cas los xij, lwm txoj hauv kev no yog siv sijhawm ntev thiab tuaj yeem qhia tus neeg tshawb xyuas yuam kev.

Ib txoj hauv kev uas pab txhawb lub hom phiaj thiab kev ntsuas ncaj qha ntawm neurite fragmentation suav nrog kev siv Analyze Particles algorithm ntawm ImageJ los ntsuas thaj tsam ntawm neurite fragments hauv micrographs ntawm kab lis kev cai. Cov kev ntsuas no yog siv los xam qhov Degeneration Index (DI), qhov ntsuas nruab nrab ntawm {{0}}.0 thiab 1.0 cuam tshuam txog qib ntawm neurite degeneration (Sasaki li al., 2009).

Thaum DI kev soj ntsuam tuaj yeem siv los txheeb xyuas cov qib sib txawv ntawm neurite fragmentation, qhov tseeb ntawm kev ntsuas feem ntau raug txwv los ntawm ntau qhov chaw ntawm kev ua yuam kev, thiab kev siv cov txheej txheem tau txwv rau kev cog qoob loo lossis cov hlwb loj hlob hauv cov khoom siv microfluidic compartmentalized. Ntxiv mus, DI tsom xam yog siv sij hawm-siv thaum extrapolated rau cov duab loj. Yog li, peb tau nrhiav kom kov yeej cov kev txwv no los ntawm kev hloov kho DI txheej txheem kev tshuaj ntsuam xyuas kom yooj yim dua qhov raug thiab ua haujlwm tau zoo, thaum nthuav dav.

Hauv txoj kev tshawb fawb tam sim no, peb txheeb xyuas ntau qhov kev ua yuam kev uas feem ntau cuam tshuam nrog DI kev tshuaj ntsuam xyuas thiab tshaj tawm cov txheej txheem kev hloov kho uas txo qis ntau yam kev ua yuam kev. Peb suav nrog cov txheej txheem txhim kho hauv ImageJ macro hu ua Automated Neurite Degeneration Index (ANDI) kom ua tiav DI kev tshuaj ntsuam xyuas los ntawm cov duab loj. Kev ua haujlwm ntawm ANDI Macro tsuas yog xav tau software thiab cov ntaub ntawv uas qhib thiab muaj pub dawb. Kev ntsuas tau los ntawm kev siv ANDI tau txais txiaj ntsig los ntawm kev ua kom raug ntau ntxiv thaum txo cov sij hawm xav tau los ua cov tshuaj ntsuam xyuas.

Ntxiv mus, peb ua qauv qhia yuav ua li cas txoj kev tshuaj ntsuam tshiab tuaj yeem siv los ntsuas qhov tseeb thiab nrawm ntawm neurite degeneration hauv cell kab lis kev cai qauv ntawm PD uas cuam tshuam nrog cov hlwb uas tsis muaj kev sib cais. Sib sau ua ke, cov txheej txheem kev txhim kho no tuaj yeem ua rau muaj kev pom tshiab rau hauv cov xwm txheej hauv qab txheej txheem cellular tseem ceeb cuam tshuam nrog neurodegeneration.

Cov ntaub ntawv thiab cov txheej txheem

Cell kab lis kev cai

Kab lis kev cai ntawm Lund human mesencephalic (LUHMES) hlwb tau tsim nyob rau hauv Lab Tek II yim-zoo chamber slides coated nrog poly-L-ornithine (PLO) thiab fibronectin, poly-D-lysine (PDL) thiab laminin, tag nrho plaub substrates, los yog tsis muaj. substrates, raws li tau hais tseg. Txhawm rau coated nrog PLO thiab fibronectin, slides tau incubated thaum hmo ntuj ntawm chav tsev kub nrog ib tug tov uas muaj 200 mg / ml PLO (Sigma-Aldrich, catalog #P3655).

Tom qab ntxuav nrog dej tsis muaj menyuam (Avantor), cov swb tau tsim nrog 2 mg / ml fibronectin (Sigma-Aldrich, catalog #F0895) rau 3 h ntawm 37 degree. Tom qab ntawd cov slides raug ntxuav nrog tsis muaj menyuam PBS (Corning Life Sciences, catalog #21-040-CV) thiab tso cai rau huab cua qhuav. Txhawm rau coated nrog PDL thiab laminin, slides tau incubated nyob rau hauv chav tsev kub nrog ib tug tov uas muaj 100 ng / ml PDL (Sigma-Aldrich, catalog #P7280) thiab 10 mg / ml laminin (Corning, catalog #354232).

Tom qab ib hmo incubation, cov slides tau ntxuav nrog tsis muaj menyuam PBS thiab tso cai rau cua qhuav. Txhawm rau tiv thaiv tag nrho plaub lub substrates, cov txheej txheem tau hais los saum toj no tau ua ua ntu zus. LUHMES hlwb (ATCC, RRID: CVCL_B056) tau plated nyob rau hauv lub coated chamber slides ntawm qhov qhia qhov ntom ntawm qhov sib txawv nruab nrab uas muaj DMEM/F12 (Invitrogen, catalog #11330057) ntxiv nrog 2 mM glutamine (VWR), 1% (v/v) N-2 ntxiv, 1 mg/ml tetracycline (Sigma-Aldrich, catalog #87128-25G), 1 mM N6, 29-Odibutyryladenosine cAMP (db-cAMP ; Enzo Life Sciences, catalog #BML-CN125-0100), thiab 2 ng/ml glial cell line-derived neurotrophic factor (GDNF; R&D Systems, catalog #212-GD-010).

Tshwj tsis yog qhov uas tau hais tseg, txhua kab lis kev cai tau plated ntawm qhov ntom ntawm 115,000 cov cell ib qhov dej. Ib qho 50% ntim tawm hloov pauv tau ua txhua 48 teev kom txog thaum kho. Txhawm rau tiv thaiv caj ces drift, cell stocks uas tau dhau mus rau rau lossis tsawg zaus tau siv rau txhua qhov kev sim.

Cov kev sim uas cuam tshuam nrog cov kab lis kev cai tseem ceeb ntawm kev sib koom siab neurons tau pom zoo los ntawm Pawg Saib Xyuas Tsiaj thiab Siv Khoom ntawm Eastern Kentucky University lossis University of Virginia. Superior cervical ganglia raug txiav tawm los ntawm hnub tom qab yug menyuam 0–6 C56Bl/6 nas, incubated ntawm 37 degree nyob rau hauv ib tug enzymatic dissociation tsis, thiab ntxuav nrog loj hlob xov xwm kom tshem tawm cov proteolytic enzymes thiab triturated.

Cell suspensions raug plated ntawm qhov ceev ntawm 7000 cell ib qhov dej hauv Lab Tek II yim-qhov chamber slides. Media tau hloov nyob rau hnub hauv vitro (DIV) 1, ua raws li kev hloov xov xwm txhua hnub. Media tau ntxiv nrog cytosine arabinofuranoside (Ara-C, Millipore-Sigma) ntawm DIV1-DIV3 los tiv thaiv kev loj hlob ntawm cov hlwb uas tsis yog neuronal, tom qab ntawd tshem tawm Ara-C los ntawm kev tshaj tawm tsawg kawg 24 teev ua ntej kev kho mob raws li tau hais tseg. Rau kev sim ua nyob rau hauv Eastern Kentucky University uas muaj kev kho hydrogen peroxide, chamber slides tau coated nrog 200 mg / ml PLO (Sigma-Aldrich, catalog #P3655), 2 mg / ml fibronectin, 100 ng / ml PDL (Sigma-Aldrich, catalog #P7280) thiab 10 mg/ml laminin (Corning, catalog #354232) ua ntej siv, thiab dissociation tau ua los ntawm 30-min incubation nyob rau hauv tsis muaj DMEM (ThermoFisher, catalog #11960- 044) muaj 0.025% trypsin (Corning Life Sciences, catalog #25-053-CI) thiab 0.3% collagenase (Worthington Biochemical, catalog #LS004176).

Kev loj hlob tawm rau cov kev sim no muaj DMEM ntxiv nrog 10% fetal bovine serum (Invitrogen, catalog #26140-079), 2 mM L-glutamine (Corning, catalog #25-005-CI), thiab 40 ng/ml paj hlwb loj hlob (Fisher Scientific, catalog #13-257-019); 5 mM Ara-C (Sigma-Aldrich) tau siv los tshem tawm cov hlwb uas tsis yog neuronal.

Rau kev sim ua nyob rau hauv University of Virginia koom nrog kev tshem tawm NGF, cov hlwb raug plated rau ntawm chav tsev slides coated nrog 100 ng / ml PDL (Millipore Sigma) thiab 10 mg / ml laminin (Invitrogen), thiab dissociation tau ua los ntawm 20-min incubation nyob rau hauv tsis muaj xws li DMEM / F12 muaj BSA (10 mg / ml, Sigma-Aldrich), collagenase hom 2 (4 mg / ml, Worthington Biochemical), deoxyribonuclease 1 (Millipore Sigma), thiab hyaluronidase (Sigma-Aldrich). Kev loj hlob xov xwm rau cov kev sim no muaj DMEM ntxiv nrog 10% fetal bovine serum (Invitrogen), 1 U / ml penicillin / streptomycin (Invitrogen), thiab 45 ng / ml ntawm NGF (purified los ntawm salivary qog); 10 mM Ara-C tau siv los tshem tawm cov hlwb uas tsis yog neuronal.

Kev kho mob ntawm tes

Cov hlwb sib txawv ntawm LUHMES tau raug pom los ntawm qhov qhia tau hais tias muaj ntau ntawm {{{0}}}hydroxydopamine (6-OHDA; Sigma-Aldrich, catalog #162957) los yog lub tsheb tov rau hnub thib tsib hauv qhov sib txawv nruab nrab. Cov khoom lag luam ntawm 6- OHDA tau npaj rau hauv chilled PBS uas muaj 0.02% ascorbate (Sigma-Aldrich, catalog #A5960), thiab aliquots tau khaws cia rau hauv lub teeb-tiv thaiv vials hauv qab roj inert ntawm 80 degree . Tom qab 24 teev ntawm kev kho mob, cov hlwb raug kho los ntawm incubation hauv PBS uas muaj 4% paraformaldehyde (PFA) rau 25 min ntawm chav tsev kub.

Txhawm rau txiav txim siab lub luag haujlwm ntawm c-Jun N-terminal kinase (JNK) hauv neurite degeneration tshwm sim los ntawm oxidative kev nyuaj siab, kab lis kev cai raug pretreated rau 1 h nrog 10 mM SP600125 (Sigma-Aldrich, catalog #S5567) los yog tsheb tov ua ntej ua tiav kev hloov pauv ntawm cov xov xwm nrog cov xov xwm uas muaj 7.5 mM 6-OHDA, 7.5 mM 6-OHDA thiab 10 mM SP600125, los yog cov kev daws teeb meem tsheb. Cov khoom lag luam ntawm SP600125 tau npaj los ntawm kev rhuav tshem cov inhibitor hauv sterile dimethylsulfoxide (DMSO), thiab khov aliquots tau muab cia rau hauv lub teeb-tiv thaiv vials uas muaj cov roj inert ntawm 20 degree. Kev kho mob tau ua xws li cov hlwb raug tsis pub ntau tshaj 0.1% DMSO.

Thawj kab lis kev cai ntawm sympathetic neurons raug kho ntawm DIV5. Rau cov kev sim uas muaj cov neurite degeneration induced los ntawm hydrogen peroxide, cov kab lis kev cai tau raug rau 500 mM hydrogen peroxide (Sigma-Aldrich, catalog #H1009-100ML) los yog lub tsheb tov uas muaj sterile dej (Cytiva, catalog #SH30529.02) rau 24h.

Rau cov kev sim uas cuam tshuam nrog kev tshem tawm NGF, SCG kab lis kev cai tau txais kev hloov pauv nrog cov xov xwm loj hlob lossis raug NGF deprivation. SCG kab lis kev cai yuav tsum tau muab tshem tawm ntawm NGF tau muab yaug peb zaug nrog NGF-dawb kev loj hlob xov xwm thiab tom qab ntawd incubated hauv NGF-dawb loj hlob xov xwm rau 80 teev yam tsis muaj kev hloov xov xwm. Tom qab kev kho mob, cov hlwb raug kho los ntawm incubation hauv 4% PFA hauv PBS rau 30 feeb, tom qab ntawd tshem tawm PFA los ntawm kev yaug nrog PBS.

Staining thiab fluorescence microscopy

Rau kev sim nrog LUHMES hlwb, nrog rau cov kev sim uas muaj kev sib koom siab neurons kho nrog hydrogen peroxide, yim-zoo chamber slides uas muaj cov hlwb ruaj khov tau permeabilized nrog 0.1% Triton X-100 hauv PBS thiab thaiv rau 1 h nrog 10% ib txwm tshis serum hauv PBS uas muaj 0.1% Triton X-100. Tom qab ntawd cov slides tau incubated nrog ib tug thawj antibody tshwj xeeb rau b I tubulin (Covance; 1:1000, RRID: AB_2313773) nyob rau hauv ib tug thaiv kev daws ntawm 4 degree thaum hmo ntuj. Tom qab ntawd, cov slides tau ntxuav nrog PBS, incubated nrog ib tug thib ob antibody ua ke rau Alexa Fluor 488 (Thermo Fisher Scientific; 1:500, RRID: AB_142495) rau 90 min ntawm chav tsev kub, thiab raug ntxuav ntxiv.

Rau cov kev sim uas muaj tyrosine hydroxylase (TH) staining, cov slides tau raug rau ib qho ntxiv incubation nyob rau hauv PBS uas muaj 10% tshis serum, 0.1% Triton X-100, antibody tshwj xeeb rau TH (Sigma- Aldrich, 1:400, AB152) ntawm 4 degree thaum hmo ntuj. Cov slides raug ntxuav nrog PBS, incubated nrog ib tug thib ob antibody ua ke rau Alexa Fluor 568 (Thermo Fisher Scientific; 1:500, RRID: AB_1500889), thiab raug rau ntxiv PBS ntxuav.

Tom qab ua tiav cov tshuaj tiv thaiv kab mob rau b III-tubulin lossis TH, cov swb tau tsim nrog 5 mg / ml 49, 69-diamidino-2-phenylindole (DAPI; catalog #D9542) hauv PBS rau 5 feeb. Tom qab ntawd cov swb tau ntxuav nrog PBS, thiab cov ntawv npog tau txuas nrog Fluoromount G mounting nruab nrab (Southern Biotech, catalog #0100-01).

Rau cov kev sim uas cuam tshuam nrog cov neurons sympathetic neurons raug NGF tshem tawm, cov slides raug thaiv rau 1 h los ntawm incubation ntawm chav tsev kub nyob rau hauv ib tug tsis muaj 5% ib txwm tshis serum (Lub neej Technologies) thiab 0.0005% Triton X-100 hauv PBS, ua raws li incubation hauv thawj cov tshuaj tiv thaiv kab mob uas muaj nas los tiv thaiv nas Tuj1 (1:1000, Covance) hmo ntuj ntawm 4 degree . Tom qab ntawd, cov slides tau muab yaug nrog PBS, incubated rau 1 h nyob rau hauv tshis anti-nas IgG Alexa Fluor 488 (1:800, Invitrogen) thiab 0.1 mg / ml Hoechst 33342 (Invitrogen) nyob rau hauv thaiv tsis pub, raug rau ntxiv PBS tso cai yaug, thiab kom qhuav. Cov slides tau teeb tsa nrog Fluoromount-G (Southern Biotech, catalog #0100-01) thiab khaws cia hauv chav sov.

Slides uas muaj LUHMES hlwb tau pom nrog 20 lub hom phiaj ntawm Nikon Eclipse II Ti-U Inverted Microscope System nrog Lumencor Mira 4-Channel LED teeb qhov chaw. 1280 1024-pixel duab raug ntes siv Nikon DSQi1Mc 5 MP CCD Lub Koob Yees Duab thiab NIS Elements 3.22.15 (Tsim 738). Tshwj tsis yog qhov uas tau hais tseg, tag nrho cov duab theem sib piv tau raug ntes siv lub teeb ci thiab lub sij hawm raug teeb tsa uas tsim qhov nruab nrab ntawm qhov siv ntawm 165 hauv 8-ntsis duab.

Txhawm rau txo qis tom qab shading lossis lwm yam tsis sib xws, ua ntej txhua qhov kev sib tham microscopy, ib daim duab ntawm lub qhov dej khoob tau raug ntes thiab siv rau kev kho keeb kwm yav dhau thaum LUHMES cell imaging. Slides uas muaj kev sib koom siab neurons kab lis kev cai ntawm Eastern Kentucky University lossis University of Virginia tau thaij duab siv Zeiss LSM 800 microscope system lossis Zeiss LSM 980 microscope system, raws li. Cov duab raug ntes ntawm 20 qhov loj thiab 1024 1024 daws teeb meem siv Zen software (Carl Zeiss Microscopy).

Kev tsom xam ntawm cell viability

Sib txawv LUHMES kab lis kev cai ntawm tes tau tsim nyob rau ntawm Lab Tek II yim-zoo chamber slides uas tau coated raws li qhia nrog ntau yam substrates. Kev hloov pauv 50% kev tshaj tawm tau ua txhua 48 teev, thiab cov hlwb raug kho nrog 4% PFA hauv PBS rau hnub thib rau ntawm kev sib txawv. Slides uas muaj cov hlwb ruaj khov tau raug staining nrog DAPI, thiab cov duab ntawm nuclear staining tau ntes nrog Nikon Eclipse II Ti-U Inverted Microscope System thiab Nikon DS-Fi1 5 MP CCD Lub Koob Yees Duab.

Cells tau qhab nia zoo los yog tsis noj qab haus huv los ntawm tus kws tshawb fawb dig muag raws li qhov pom ntawm cov nuclei. Cov cell uas muaj tsawg dua thaj chaw nuclear lossis nuclear fragmentation tau qhab nia zoo li tsis zoo. Tus naj npawb ntawm cov hlwb noj qab haus huv ib daim duab tau qhab nia tsawg kawg tsib qhov kev pom ntawm ib tus mob hauv txhua qhov kev sim.

DI ntsuas

Kev ntsuas DI ib txwm siv tau siv lub pob Fiji Is Just ImageJ (FIJI) uas muaj ImageJ 1.52p los ntawm ib txoj hauv kev uas suav nrog cov ntsuas dav dav rau DI tsom xam (Shin li al., 2012; Di Stefano et al., 2015; Sasaki li al., 2016; Loreto et al., 2020; Shin thiab Cho, 2020). Yim-ntsis cov duab ntawm LUHMES cov hlwb sib txawv nrog cov axon zoo sib cais tau raug rau binarization siv lub neej ntawd binarization algorithm, nrog binary xaiv teeb tsa los tsim cov duab nrog lub hlwb dub nyob rau hauv pem hauv ntej thiab tom qab dawb.

Cov cuab yeej kos duab dawb siv tau siv los taug qab txhua thaj chaw duab uas muaj lub cev ntawm tes, thiab tag nrho cov pixels dub tau hloov mus rau dawb hauv thaj chaw taug qab. Cov cuab yeej ntsuas ntawm ImageJ tau siv los ntsuas tag nrho thaj tsam dub ntawm cov duab binarized tshwj xeeb tshaj yog cov neurites. Cov cuab yeej Analyze Particle tau siv tom qab siv qhov loj me ntawm 20–10,000 pixels thiab qhov chaw ncig ntawm 0.2–1.0 txhawm rau txheeb xyuas cov khoom tawg. Qhov summed cheeb tsam ntawm tag nrho cov neurite fragments ces muab faib los ntawm tag nrho cov dub cheeb tsam los xam DI ntawm daim duab.

Rau cov kev sim uas cuam tshuam txog kev ua kom zoo ntawm qhov loj me me rau kev txheeb xyuas cov kab mob neurite, cov txheej txheem ncig ntawm {{0}}}.2–1.00 tau siv rau txhua qhov kev ntsuas particle. Particle Remover plugin (version 20{{10}}}4/02/09), tsim los ntawm Wayne Rasband, tau txais los ntawm https:// imagej.nih.gov/. Qhov twg tau qhia, daim ntawv thov ntawm Particle Remover plugin siv qhov loj me ntawm 0-9 pixels thiab qhov tsis sib xws ntawm 0.0-1.0 tau ua kom tshem tawm cov khoom me me, uas tsis yog-neurite los ntawm binarized dluab tau los ntawm theem sib piv micrographs.

Ib yam li ntawd, qhov loj me ntawm {{0}}}–4 pixels thiab qhov tsis sib xws ntawm 0-1.0 tau siv los tshem tawm cov khoom me me los ntawm cov duab binarized tau los ntawm fluorescence micrographs sawv cev rau b III-tubulin immunostaining. Tshwj tsis yog hais tias muaj lwm yam qhia, ua ntej DI tsom xam tag nrho cov duab theem-qhov sib piv tau raug rau qhov sib txawv los ntawm kev siv ImageJ los siv lub Lookup Table (LUT) nrog qhov tsawg kawg nkaus ntawm 90 thiab siab kawg ntawm 205.

Kev ntsuas ntawm cov khoom tsim tawm fragmentation

Cov kab lis kev cai uas muaj cov kab mob LUHMES noj qab haus huv tau kho nrog 4% PFA nyob rau hnub thib rau ntawm kev sib txawv thiab immunolabeled rau b III-tubulin. Siv 20 lub hom phiaj, 1280 1024-pixel theem-qhov sib piv micrographs thiab fluorescence micrographs, txhua qhov kev piav qhia zoo ib yam ntawm qhov kev pom, tau raug ntes los ntawm cov cheeb tsam randomly. Tag nrho kaum daim duab tau raug ntes los ntawm cuaj kab lis kev cai.

ImageJ tau siv los tsim cov ntawv luam binarized ntawm thawj theem-zoo sib xws thiab fluorescence micrographs, nrog rau cov ntawv theej ntawm theem-tsis sib xws micrographs uas tau binarized tom qab kev sib piv ntawm kev siv LUT nrog qhov tsawg kawg nkaus ntawm 90 thiab siab kawg ntawm 205. Rau txhua qhov teeb tsa ntawm cov duab, tus kws tshawb nrhiav qhov muag tsis pom kev tau txheeb xyuas qhov me me, 150 150-pixel thaj tsam ntawm thawj theem-zoo sib xws micrograph uas tshwj xeeb rau kev noj qab haus huv thiab tsis zoo neurite.

Cov cheeb tsam loj thiab kev tswj xyuas tau muab khaws cia rau hauv thaj tsam ntawm kev txaus siab (ROI) tus thawj coj ntawm ImageJ thiab siv los ua DI ntsuas hauv thaj tsam sib luag ntawm cov duab binarized los ntawm tib daim duab teeb. Cov kev ntsuas DI tau ua los ntawm kev siv cov txheej txheem ib txwm muaj uas muaj qhov loj me me ntawm 20 -10, 000 pixels. Txij li tag nrho cov cheeb tsam ntsuas muaj tshwj xeeb cov neurons tsis zoo, cov khoom tawg tau pom tau suav tias yog cov khoom cuav.

ANDI Macro

ANDI Macro tau sau siv ImageJ Macro lus. Lub macro nta cov haujlwm ua haujlwm uas ua rau tus neeg siv xaiv txoj hauv kev rau peb phau ntawv qhia, ib phau ntawv muaj cov duab ntawm cov neurons immunolabeled rau cov cim neuronal xws li b III-tubulin, ib phau ntawv thib ob uas muaj cov duab sib xws uas piav qhia txog nuclear staining, thiab peb phau ntawv teev npe. tsim los txuag cov ntaub ntawv tau. Cov cheeb tsam fragment thiab tag nrho cov cheeb tsam neurite ntawm cov duab hauv cov npe xaiv tau raug xam los ntawm kev ua haujlwm ntawm ImageJ. Nyob rau hauv luv luv, ib daim duab piav qhia txog nuclear labeling yog qhib, raug rau kev sib piv, thiab binarized. Particle tshem tawm plugin yog siv los tshem tawm cov khoom me me, uas tsis yog-nuclear artifacts los ntawm cov duab binarized, thiab cov duab binarized yog raug rau ntau qhov sib txawv thiab yaig.

Ib daim duab sib thooj ntawm cov hlwb immunolabeled rau b III-tubulin (los yog lwm yam neuronal marker) raug qhib thiab raug rau qhov sib txawv. Daim duab binarized uas piav qhia txog cov cell nuclei loj yog ces rho tawm ntawm daim duab uas muaj cov hlwb immunolabeled rau b III-tubulin, yog li tsim cov duab uas muaj cov neurite nkaus xwb. Cov duab neurite yog tom qab ntawd binarized thiab raug tshem tawm ntawm qhov nruab nrab ntawm 0 thiab 4 pixels. Neurite fragment cheeb tsam yog xam los ntawm kev siv lub Analyze Particle algorithm los ntsuas particles nrog ib ncig ntawm 0.2 thiab 1.0 thiab loj ntawm 5 thiab 10, 0000 pixels. Tag nrho cheeb tsam neurite yog xam los ntawm kev siv lub ntsuas ntsuas los ntsuas tag nrho cheeb tsam dub. Ib daim duab piav qhia txog thaj chaw binarized neurite nrog cov ntsiab lus tseem ceeb tau khaws cia rau hauv phau ntawv teev npe los ntawm tus neeg siv, nrog rau .csv cov ntaub ntawv qhia txog thaj chaw neurite thiab tag nrho cov neurite cheeb tsam ntsuas.

Ntxiv nrog rau cov haujlwm uas tau hais los saum no, ntau qhov kev ua haujlwm ntxiv tau suav nrog hauv macro los txhawb kev yooj yim ntawm kev siv thiab txwv cov neeg siv yuam kev. Cov no suav nrog cov haujlwm uas tshem tawm cov ntaub ntawv teev teev los ntawm cov duab thiab xyuas kom meej tias kev ntsuas tau txais hauv pixel units; teeb tsa ImageJ xim teeb tsa thiab binary xaiv rau tsim cov duab rho tawm thiab binarization; txheeb xyuas tias cov npe xaiv rau cov duab ntawm b III-tubulin staining thiab DAPI staining muaj cov duab sib npaug; kaw cov duab yav dhau los siv los txhim kho lub sijhawm ua haujlwm; meej cov txiaj ntsig los ntawm kev txheeb xyuas yav dhau los ua ntej kev ua haujlwm ntawm kev tshuaj ntsuam tshiab; thiab tiv thaiv qhov tsis raug cuam tshuam nrog .ini cov ntaub ntawv tsim los ntawm Windows 10.

Rau cov kev sim uas cuam tshuam nrog kev txheeb xyuas ntawm cov paj hlwb muaj txiaj ntsig, ANDI Macro tau hloov kho kom suav nrog kev ua haujlwm uas nthuav tawm lub thawv sib tham ua rau tus neeg siv los tswj cov sij hawm uas binarized dluab ntawm nuclei yuav dilated. Lub version ntawm ANDI featuring qhov kev hloov kho no yog hu ua ANDI v1.1. Hloov tus nqi hauv lub thawv sib tham mus rau tus lej siab dua ua rau qhov loj me ntawm soma tshem tawm thaum hloov tus nqi mus rau tus lej qis dua txo qhov kev tshem tawm soma. Lub neej ntawd tus nqi yog 12, uas yog tus naj npawb ntawm dilations siv rau txhua qhov kev sim nrog LUHMES hlwb. Tus nqi ntawm 16 tau siv los tshem tawm cov cheeb tsam ntawm lub cev loj dua thaum kev soj ntsuam ntawm cov paj hlwb sympathetic.

ANDI Macro yog qhov qhib thiab muaj pub dawb rau siv lossis hloov kho ntawm qhov URL hauv qab no: https://github. com/kraemerb/kaemerlab. Kev ua haujlwm raug ntawm ANDI tau raug txheeb xyuas siv ntau lub khoos phis tawj tus kheej uas muaj Windows 10 operating system. Ib daim duab teeb uas muaj LUHMES kab lis kev cai ntawm tes uas nthuav tawm ntau qib ntawm neurite degeneration kuj muaj nyob rau ntawm qhov chaw nyob saum toj no thiab tuaj yeem siv los tshawb xyuas qhov tseeb ntawm macro los ntawm lwm hom khoos phis tawj. Lub vev xaib chaw nyob kuj tseem muab kev nkag mus rau cov txheej txheem piav qhia txog kev siv macro.

Lub sij hawm kwv yees

Txhawm rau ntsuas lub sij hawm yuav tsum tau ua manually ua DI ntsuas, theem-qhov sib piv micrographs qhia txog LUHMES hlwb uas tau kho nrog lub tsheb, 2.5 mM 6-OHDA, 5.0 mM 6-OHDA, los yog 7.5 mM 6-OHDA tau raug DI cov qhab nia siv cov txheej txheem uas tau tsim ua ntej (Kraemer li al., 2014). Txhua daim duab tau raug ua kom zoo sib xws thiab binarization. Cov cuab yeej pub dawb ntawm ImageJ tau siv los ua digitally tshem tawm txhua lub cev ntawm tes los ntawm cov duab binarized. Cov cuab yeej tsom xam Particle tau siv los ntsuas cov khoom tawg nrog ib ncig ntawm 0.2 thiab 1.0 thiab qhov loj ntawm 20 thiab 10,{18}} pixels, thiab cov cuab yeej ntsuas tau siv los txiav txim tag nrho thaj chaw neurite.

Cov txiaj ntsig ntawm thaj chaw neurite tawg thiab tag nrho thaj chaw neurite raug xa mus rau ib daim ntawv nthuav dav hauv Microsoft Excel, thiab kev ua lej hauv Excel tau siv los suav cov qhab nia DI. Tom qab qhib daim duab nrog ImageJ, lub sijhawm yuav tsum tau ua manually ua txhua yam ntawm cov txheej txheem no tau ntsuas. Lub sijhawm ntsuas nruab nrab tau suav los ntawm 36 tag nrho cov duab. Cov duab tau txais los ntawm cuaj txoj kab lis kev cai ywj pheej ntawm tes, thiab kev tshuaj ntsuam tau ua los ntawm kev siv ib daim duab rau ib qho kev kho mob los ntawm txhua qhov kev npaj kab lis kev cai ntawm tes. Txhawm rau txiav txim siab qhov nrawm ntawm DI kev ntsuas tau ua los ntawm ANDI Macro, tib cov duab teev tau siv los txiav txim siab lub sijhawm yuav tsum qhib macro hauv ImageJ, khiav cov haujlwm macro, hloov cov ntaub ntawv mus rau Excel spreadsheet, thiab xam cov DI.

Kev txheeb cais

Kev sib piv ntau yog nthuav tawm raws li lub thawv thiab lub thawv ntawv, qhov nruab nrab kab sawv cev rau qhov nruab nrab, cov thawv txuas mus rau 25th thiab 75th feem pua, thiab whiskers qhia txog 5th thiab 95th feem pua. Cov ntsiab lus tau suav nrog hauv GraphPad Prism 9.1.2 los ntawm kev ntsuas rov qab ANOVA nrog kev sib piv tom qab kev sib piv siv Sidák kho. Qhov zoo ib yam rau ntau cov ntaub ntawv tau sim siv Shapiro-Wilk xeem hauv IBM SPSS 26.

Thaum lub assumption ntawm normality tau ua txhaum (Fig. 1C, 3D), pab pawg neeg sib piv tau ua los ntawm cov nonparametric Friedman xeem nrog Dunn qhov kev xeem ua rau post hoc sib piv. Cov kev txheeb xyuas kev sib raug zoo tau piav qhia raws li cov duab kos thiab suav nrog cov kab uas haum zoo tshaj plaws. Cov kev sib raug zoo no tau txheeb xyuas siv Pearson cov khoom lag luam-lub sijhawm sib raug zoo lossis Spearman's r thaum qhov kev xav ntawm qhov qub raug ua txhaum cai. Qhov zoo ib yam ntawm cov seem rau kev txheeb xyuas kev sib raug zoo raug soj ntsuam los ntawm kev tshuaj xyuas qhov muag ntawm QQ cov phiaj xwm sib thooj.

Pearson cov khoom lag luam-lub sijhawm sib raug zoo coefficients tau muab piv nrog ib leeg siv Steiger's asymptotic z-test (Steiger, 1980; Lee and Preacher, 2013). Tsis muaj outliers raug cais tawm. Kev txheeb xyuas qhov tseem ceeb tau txais ntawm p, 0.05. P-tus nqi raug muab sib npaug rau plaub qhov chaw lej thiab tshaj tawm thoob plaws hauv cov ntawv tseem ceeb. Qhov ntau thiab tsawg thiab 95% kev ntseeg siab ib ntus kuj tau tshaj tawm rau kev soj ntsuam parametric, nrog cov txiaj ntsig ntau thiab tsawg tau piav qhia thoob plaws hauv cov ntawv tseem ceeb thiab kev ntseeg siab nyob rau hauv Table 1.

Cov ntaub ntawv muaj

Cov ntaub ntawv ntxiv txog tsab xov xwm tam sim no yuav muab qhia tawm raws li qhov kev thov tsim nyog.

Code nkag tau yooj yim

Cov cai / software tau piav qhia hauv daim ntawv yog muaj dawb hauv online ntawm https://github.com/kraemerb/kraemerlab. Cov cai muaj nyob rau hauv Extended Data 1.

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